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WO2020234170A1 - Production de sphéroïde cellulaire dans un système de culture cellulaire 2d - Google Patents

Production de sphéroïde cellulaire dans un système de culture cellulaire 2d Download PDF

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Publication number
WO2020234170A1
WO2020234170A1 PCT/EP2020/063659 EP2020063659W WO2020234170A1 WO 2020234170 A1 WO2020234170 A1 WO 2020234170A1 EP 2020063659 W EP2020063659 W EP 2020063659W WO 2020234170 A1 WO2020234170 A1 WO 2020234170A1
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Prior art keywords
cells
cell
cell culture
spheroids
specifically
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Sylvia NÜRNBERGER
Marian FÜRSATZ
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Medizinische Universitaet Wien
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Medizinische Universitaet Wien
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates

Definitions

  • the present invention relates to the field of cell culture systems.
  • EP2617807A1 discloses methods of generating spheroids using culture substrates having a plurality of dents forming compartments in which cells are cultured to form spheroids.
  • the culture substrate surface of the dent is a non-flat surface, specifically it is a smooth concave plane.
  • the dents are formed by laser irradiation on the culture substrate surface and are placed very close together, leaving almost none of the original surface. At the peripheries of the dents, the synthetic resin material of the culture substrate is melted and piled up to form banks.
  • W02017/142410A1 discloses methods of growing spheroids using a round- bottom, optically clear insert plate, which can be reversibly attached to a
  • the cells are cultured in the wells of the insert plate, wherein the bottom of each well has a concave arcuate surface (see Fig. 3).
  • EP3296018A1 discloses methods of growing arrays of organoids using a cell culture device, which has a surface imprinted with cavities or microwells of various sizes, shapes and depths.
  • the wells are round (U) -bottomed, so that the seeded cells are all gathered at the bottom of the cavities.
  • US2016/0324991A1 discloses micronized platforms comprising a plurality of testing regions, which may be individual chambers holding tumor spheroids, for in vivo implantation in an animal host.
  • the platform is used to study biosamples in an in vivo setting.
  • US2019/0055590A1 discloses methods of generating spheroids, which are useful for screening techniques, using multi-well plates, wherein each of the wells has a lowly absorptive bottom having a U-shaped section.
  • EP2759592A1 discloses cell culture devices comprising suspension chambers called culture spaces (see e.g., Fig 3), wherein the surface of each of the culture spaces is processed by glass processing or by forming a functional group thereon by plasma treatment, so that the surface has a water contact angle of 45 degrees or less, i.e. it is a hydrophilic surface.
  • cell culture devices used for the generation of spheroids have a concave bottom, to ensure that cells can aggregate, and/or are anti-adhesive in the hopes of promoting generation of spheroids by preventing cells from sticking to the bottom of the device.
  • a method of producing three- dimensional spheroids of cells using a two-dimensional cell culture device comprising at least one chamber having an adhesive, flat bottom growth surface, which is separated by incisions into compartments, allowing distribution of cell culture media across said at least one chamber, comprising the following steps: i. introducing cell culture media comprising cells into the cell culture device, ii. allowing said cells to settle within the compartments, and
  • cell spheroids are formed in 80, 81, 82, 83, 84, 85, 86, 87, 88,
  • the compartments on the bottom surface of the two-dimensional cell culture device described herein are separated by incisions or by elevated structures, specifically bulges.
  • the incisions or bulges described herein are formed by a laser or a sharp object.
  • the bulges described herein are bulges comprising incisions.
  • cell spheroids are formed in at least 80% of the compartments and in at least 50, 60, or 70%, preferably at least 80 or 90%, of the incisions or bulges described herein.
  • the cells are mammalian cells or invertebrate cells.
  • the cells described herein are human cells, preferably human cells derived from cartilage or fat tissue.
  • the cells used in the method described herein are adipose derived stromal/stem cells (ASC) or human articular chondrocytes (HAC).
  • ASC adipose derived stromal/stem cells
  • HAC human articular chondrocytes
  • the method described herein comprises co culturing cells, specifically at least two different kinds of cells.
  • the co cultured cells used in the method described herein are ASCs and human articular chondrocytes HACs.
  • two different cell types are cultured in a ratio, for example at a ratio of 50:50 or 20:80 in the two-dimensional cell culture device described herein.
  • ASCs and HACs are co-cultured in the cell culture device described herein, they are preferably cultured in a ratio of 80:20, 50:50 or 20:80% ASC:HAC.
  • the compartments described herein comprise a diameter of at least 0.1 mm and up to 15 mm. Specifically, the compartments described herein comprise a diameter of at least 0.2, 0.3, 0.4, 0.5,
  • the compartments are of a regular shape, such as an angular shape, for example square, rectangular, triangular, regular pentagon, regular hexagon, regular octagon, or a round shape, for example circular or oval.
  • the compartments are of an irregular shape, having sides and/or angles of different length and size, for example irregular pentagon, irregular hexagon, or irregular octagon.
  • the two-dimensional cell culture device described herein comprises at least one chamber.
  • the two-dimensional cell culture device described herein comprises at least 2 chambers, specifically at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
  • the at least one chamber described herein comprises a diameter of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140 or 150 mm.
  • the cell culture device described herein may comprise chambers of different sizes and shapes.
  • the chambers described herein may comprise compartments of different sizes and shapes.
  • the cells are cultured in the cell culture device described herein for at least a day, specifically 3 days, preferably at least 1 week. Specifically, the cells are cultured according to the method described herein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or longer. Specifically, the cells are cultured for at least 1, 2, 3 or 4 weeks. Specifically, the cells are cultured in cell culture media under conditions allowing cell growth, specifically under appropriate temperature and gas mixture. Specifically, the cells are cultured at a temperature between 35 ° C and 38 C, specifically about 37 C. Specifically, the cells are cultured at about 5%
  • the cells are typically cultured under the appropriate oxygen concentration. Cells may also be cultured under hypoxic conditions wherein 0 2 is comprised at a concentration of less than 20 %.
  • the cell spheroids described herein are generated after culturing cells in the cell culture device described herein for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days or longer. Specifically, the cell spheroids are generated after about 1, 2, 3, 4, 5 or 6 weeks.
  • the method described herein comprises the step of harvesting the cell spheroids.
  • the cell spheroids are harvested after culturing in the cell culture device for at least 1, 2, 3, 4, 5 or 6 days or 1, 2, 3 or 4 weeks.
  • the cell spheroids produced by the method described herein are of uniform size which correlates to the size of the compartments within the, at least one, chamber of the cell culture device described herein. Specifically, the size of a cell spheroid corresponds to the surface area of the corresponding
  • cell spheroids of uniform size are formed in at least 80%, preferably at least 90% of the respective compartments.
  • cell spheroids of uniform size are produced according to the method provided herein in at least 80, 81, 82,
  • cell spheroids of uniform size are formed in at least 50, 60, or 70%, preferably at least 80 or 90%, of the incisions or bulges described herein.
  • the cell spheroids produced according to the method described herein are cultured in a three-dimensional cell culture comprising a hydrogel and/or a scaffold.
  • the three-dimensional cell culture comprises a fibrin hydrogel, specifically high and/or low-density fibrin hydrogel.
  • the cell spheroids produced according to the method described herein are used for regenerative medicine applications, such as in vitro production of cartilage, bone or muscle tissue.
  • the cell culture device provided herein is used for high- throughput screening of exogenous influences of physical, chemical or biological nature.
  • the cell culture device can be used to screen the effect of chemical compounds or environmental conditions on cell spheroids.
  • Figure 2 Bright-field and fluorescence imaging of pellet formation for ASC and 0.5:0.5 ASC:HAC co-culture on a 3mm grid plate over a period of 29 days.
  • Figure 3 SEM imaging of different stages during pellet formation (a) Beginning contact loss and retraction at grid borders (b) Rolling up of cell layer (c) Further aggregation and formation of a knob-like structure (d) Fully formed pellet (pbar: 200 pm) [0036]
  • FIG. Histological sections of different ASC:HAC ratio pellets grown on 3 mm grid plates, stained for ECM formation (Azan), collagen 2 and GAGs (Alcian). (pbar: 100 pm; pbar inserts: 20 pm)
  • Figure 7 Sprouting behavior of ASC pellets (lmm and 3mm) embedded into low and high-density fibrin, and 0.5:0.5 ASC:HAC pellets (3mm) embedded in low density fibrin.
  • Figure 8 Bright-field and corresponding histological sections of lmm and 3mm ASC pellets in low and high-density fibrin and 3mm 0.5:0.5 ASC:HAC co culture pellets in low density fibrin after 14 days of culture (pbar bright-field:
  • Figure 11 C2C12 cells spreading over the compartment and forming nodules within a non-confluent cell layer.
  • FIG. 12 Macroscopic images of the grid plate with 3mm grid size (left) and a standard petri dish with flat surface (right).
  • Figure 13 SEM image of C02-laser generated incisions within the plastic surface of the cell culture device. Magnification 150x.
  • Figure 14 SEM images of plastic surfaces compartmented with three different instruments (left: top view, length indicator: 400pm; right: cross section, length indicator: 100pm). Upper row: Scalpel incisions, middle row: Stanley knife, lower row: fraise. [0047] Figure 15. Bright field images of a scalpel-incised compartmentation of the growth surface with a cell layer of ASC/TERT1. Left: After seven days the cells align (arrow) and aggregate (arrow head) along the incision border. Right: After fourteen days a detachment of the cell layer from the corners is visible.
  • the bottom surface of two-dimensional cell culture devices such as for example conventional cell culture dishes, is compartmentalized and cells cultured thereon spontaneously and autonomously self-assemble into three-dimensional cell spheroids.
  • the cell culture device described herein is designed to be easily mass-producible and to reduce media consumption since numerous cell spheroids can be produced within one chamber. Accordingly, reagent cost and handling time are significantly reduced.
  • the term“three-dimensional spheroids of cells”, unanimously used with “cell spheroids”, as used herein refers to aggregates of cells in culture that comprise three-dimensional architecture.
  • the cell spheroids produced by the method described herein are of uniform size, wherein the size corresponds to the size of the compartments and/or the size of the incisions or bulges separating the compartments. It thus follows, that the size of the cellular spheroids described herein can be controlled by the size of the compartments and/or the incisions or bulges. Specifically, varying the size of the compartments allows producing cell spheroids of a pre-determined size and/or shape.
  • the cell spheroids described herein do not comprise any supplementary material such as a synthetic matrix.
  • Cellular spheroids represent excellent models for the in vitro study of biological functions of cells, including stem cells and cancer cells, specifically since they allow closely mimicking in vivo behavior.
  • cell spheroid encompasses organoids.
  • an organoid is a collection of organ-specific cell types that develops from stem cells or organ progenitors and self-organizes into a cellular spheroid.
  • organoids self-organize their structure through three- dimensional spatial arrangement and spatially restricted lineage commitment in a manner similar to in vivo.
  • cell spheroids described herein are not limited in size.
  • Exemplary, cell spheroids described herein comprise as few as 2 cells or up to lxlO 6 or more cells.
  • a cell spheroid produced by the method described herein comprises about lxlO 3 , 5xl0 3 , lxlO 4 , 5xl0 4 , lxlO 5 , 5x10 s , lxlO 6 , 5xl0 6 or lxlO 7 cells.
  • the cell spheroids described herein may be of different shape.
  • the most common shape of a cell spheroid is the round shape; however different cell types yield differently shaped cell pellets.
  • muscle cells or nerve cells form cell pellets of an elongated architecture, whereas cartilage cells typically form round cell pellets.
  • the term“cell” as used herein, is understood to refer to any cell that can be grown in a cell culture system.
  • the cells used herein are eukaryotic cells; preferably they are mammalian or invertebrate cells.
  • Mammalian cells used herein can, for example, be of primate origin, such as e.g. human, ape or monkey; rodent origin, such as e.g. mouse, rat or hamster, carnivore origin, such as e.g. cat, dog, or ungulate origin, such as e.g. cattle, horse, pig or deer.
  • mammalian cells used herein can be derived from epithelial tissue, muscle tissue, nervous tissue and/or connective tissue.
  • cells used in the method provided herein are stem cells, bone cells, muscle cells, fat cells, skin cells, nerve cells, endothelial cells, or cancer cells.
  • a single cell type is cultured or multiple cell types are cultured in the device described herein.
  • more than one, preferably two, different cell types are cultured.
  • the different cell types can be cells of the same or different species and can be cells of the same or different tissues.
  • chondrocytes, myocytes, neurons or adipocytes can be co-cultured with stem cells or cancer cells.
  • chondrocytes, myocytes, neurons or adipocytes are co-cultured with stem cells or fibroblasts.
  • the cells used in the method provided herein are stem cells, specifically adipose derived stromal/stem cells (ASCs).
  • ASCs are an excellent source for tissue regeneration, including for example regeneration of damaged cartilage, since they are easily available in high cell numbers from subcutaneous fat, and they can be harvested under reduced burden for the donor, compared to for example isolation of bone marrow derived stem cells.
  • stem cells such as ASCs, spontaneously form spheroids within about 3 to 4 weeks.
  • cell spheroids form autonomously.
  • the cells used in the method provided herein are chondrocytes, specifically human articular chondrocytes
  • HACs Chondrocytes are the only cell type present in articular cartilage and regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities.
  • chondrocytes could successfully be grown in a two-dimensional cell culture device and cell spheroids spontaneously formed by chondrocytes could be produced.
  • the produced cell spheroids were of uniform size and shape, corresponding to the size of the compartment.
  • chondrocytes grown in the cell culture device described herein formed cell pellets after 3 to 4 weeks.
  • the cells used in the method provided herein are chondrocytes and stem cells.
  • stem cells and chondrocytes are grown in a co-culture at a ratio of about 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80 or 10:90 stem cells to chondrocytes, specifically ASC:HAC.
  • the time required for formation of cell spheroids is significantly reduced when stem cells and chondrocytes are grown in co-culture in the cell culture device provided herein. While culturing adipose derived stem cells and chondrocytes separately in the cell culture device described herein, production of uniform cell spheroids is typically achieved within 3 to 6 weeks, depending on initial cell number and age of donor. Co-culture of stem cells and chondrocytes in the present cell culture device, however, yields uniform cell spheroids of
  • chondrocytes in less than 3 weeks or 2 weeks or even less than 1 week.
  • the cells cultured in the two- dimensional cell culture device described herein are invertebrate cells.
  • the invertebrate cells are cells derived from mollusks, annelids or cnidarians or arthropods, such as for example insects or arachnids.
  • the invertebrate cells used in the method described herein are derived from insects, such as for example the common fruit fly drosophila melanogaster.
  • the term“two-dimensional cell culture device” is
  • the cell culture device used herein is made of plastic or glass material or a mixture thereof.
  • the cell culture device used herein is made of a plastic material such as for example polystyrene, polypropylene or polycarbonate.
  • the most commonly used material is polystyrene, which can be colored white for example by the addition of titanium dioxide or black, for example by the addition of carbon, polypropylene is typically used for the construction of plates subject to wide changes in temperature, polycarbonate is cheap and easy to mold.
  • Commonly used two-dimensional cell culturing devices according to the method described herein include petri dishes and well plates.
  • a petri dish is a glass or plastic dish that is typically cylindrical.
  • the two-dimensional cell culture device is a petri dish it comprises one chamber.
  • said chamber is round, however there is no limitation regarding the shape of the chamber(s) of the two-dimensional cell culture device described herein. Various shapes are envisioned herein, beyond the typical round shape, such as for example square, hexagonal, rectangular or triangular chambers.
  • a well plate or also called microplate or multi-well plate, is a flat plate with multiple chambers, also called wells.
  • the chambers of multi-well plates are round or square; however various shapes are envisioned herein including for example hexagonal, rectangular or triangular wells.
  • a cell culture device as described herein comprising more than one chamber may comprise 6, 12, 24, 48, 96, 384 or 1536 chambers or even up to 3456 or 9600 chambers, preferably arranged in a 2:3 rectangular matrix.
  • a chamber of a microplate typically can hold somewhere between tens of nanoliters to several milliliters of liquid.
  • the cell culture device described herein comprises one or more chambers comprising a diameter of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or up to 150 mm or any value in between.
  • the chambers of a cell culture device as described herein comprising 6 or 12 chambers may comprise a diameter of 10, 14 or 20 mm.
  • the chambers of a cell culture device comprising 24 chambers may, for example, comprise a diameter of 10 or 13 mm
  • a device comprising 48 chambers may e.g. comprise chambers at a diameter of 6 mm
  • a device comprising 96 chambers may comprise chambers at a diameter of 5 mm.
  • the term“bottom growth surface” refers to the side of the layer at the bottom of the cell culture device which comprises the incisions and/or bulges described herein.
  • the bottom surface of the one or more chambers of the cell culture device described herein is the surface comprising the compartments that are separated.
  • the bottom surface is the upward facing side of a cell culture device whereupon the cells adhere.
  • compositions refers to fields that are separated by physical means, for example separated by incisions or bulges.
  • the compartments of the device described herein may be of various sizes and shapes. Specifically, the compartments may be squared, rectangular, round, triangular, star-shaped, hexagonal, or may be of irregular shape.
  • a chamber described herein may also comprise compartments of different sizes and/or shapes.
  • the compartments described herein comprise a diameter of at least 0.1 mm and up to 10 mm or even longer, such as for example 20, 30, 40, 50, 60, 70, 80, 90 or 100 mm. In the case, where the compartments are squared, they may comprise length and width of 0.1 to 10 mm and in the case where they are rectangular, they may comprise a length of 0.1 to 10 mm and a width of 0.1 to 10 mm.
  • the size and shape of the compartments is adjusted depending on the cell type to be cultured.
  • the compartments may be of an elongated shape, such as a rectangle comprising a length of about 1 to about 15 mm, specifically, about 2 to about 6, even more specifically about 2.5, 3, 3.5, 4, 4.5, 5,
  • the compartment comprises a length of about 2 mm and a width of about 1 mm, or a length of about 4 or 6 mm and a width of about 1 or 2 mm.
  • the compartment comprises a length of 10 or 15 mm, and a width of 0.2, 0.5 or lmm.
  • the compartments may be squared, comprising a length and a width of about 0.5 mm to 5 mm.
  • the compartments may comprise a length and a width of about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mm or more.
  • cision refers to an indentation or a cut in the bottom surface of the cell culture device described herein leading to
  • the incisions described herein can be of a straight or a curved line, preferably the incisions are in straight lines.
  • the incisions described herein are about 20 to 200 pm in depth, specifically about 50 to 150pm, specifically measured from the surface level of the bottom growth surface, preferably about 150pm, and comprise a width of about 50 to 500 pm, specifically about 50, 100, 200, 300, 400, 500 or 600 pm.
  • the incisions comprise a ridge along each edge of the incision.
  • Such ridge may comprise a height of about 40 to 100 pm or more, preferably about 50, 60, 70 or 80 pm, and a width of about 40 to 100pm pm or more, preferably about 50, 60, 70 or 80 pm.
  • the incisions are smooth.
  • the incision is below the level of the bottom surface and the ridge is above the level of the bottom surface of the cell culture device.
  • the ridge comprises overhangs.
  • the overhangs are parts of a ridge which stretch outwards from the incision and do not touch the bottom surface.
  • An overhang may comprise a width of about 10 to 40 pm or more.
  • a specific example of an incision comprising ridges is depicted in Figure 13.
  • the incisions described herein can be created using a laser, or manually using a sharp object, such as for example a scalpel, Stanley knife or a fraise.
  • a sharp object such as for example a scalpel, Stanley knife or a fraise.
  • the incisions described herein are formed using a computer- guided laser.
  • a computer-guided laser such as an argon laser or a C0 2 laser, is particularly useful since such a laser cuts the incisions with precision to create incisions of uniform dimensions comprising for example uniform depth and width.
  • the term“bulge” as described herein refers to an elevation in the bottom surface of the cell culture device described herein leading to compartmentalization of said bottom surface.
  • the bulges are of a plastic, ceramics or glass material, preferably plastic such as polystyrene or polycarbonate.
  • the bulges described herein can be of a straight or a curved line, preferably the bulges are in straight lines.
  • the bulges described herein comprise a height of at least about 40 to 200 pm, preferably about 50 to 100 pm, and a width of about 40 to 400 pm, preferably about 100 to 300 pm.
  • the bulges described herein comprise incisions and may thus be referred to as elevated incisions.
  • the bottom surface of the cell culture device described herein thus comprises incisions which are above the level of the bottom surface.
  • the cell culture device described herein comprises an adhesive bottom growth surface. Specifically, the entire bottom growth surface is adhesive, except for the incisions, bulges and/or ridges.
  • the cell culture device described herein comprises a surface modification, such as a surface coating, to facilitate cell surface adhering.
  • the device comprises no further surface modifications, except the compartmentalization described herein and such surface modifications that enable or help cells to adhere to the bottom surface of the device.
  • modification facilitating cell surface adherence may be a fibronectin coat.
  • the surface of the cell culture device, specifically the bottom surface may comprise modifications to make the surface more hydrophilic, such as for example chemical modifications or
  • the growth surface of the cell culture device comprises microstructures providing a certain roughness to the surface of the cell culture device, to aid in the formation of three-dimensional cell spheroids.
  • Such microstructures may be elevations or indentations of sub-cellular size in an irregular or regular pattern.
  • the cell culture device comprises no anti adhesive or non-adherent surface modifications that would prevent cells from adhering to the growth surface.
  • Prior art cell culture devices which are used for the formation of cell spheroids typically comprise non-adherent surfaces, so that spheroids are formed in suspension.
  • Such cell culture devices are often technically difficult to produce, as they comprise multiple elaborate separate compartments, that are intended to form individual spheroids, and comprise surface modifications, to make the surface non-adherent.
  • cells have to be seeded individually into the compartments at predefined cell numbers, making handling of the devices more difficult.
  • the cell culture device described herein is easy to produce and easy to handle.
  • the device comprises different compartments for the formation of spheroids, but these compartments are only separated by the incisions or bulges described herein, so that cell culture media is distributed evenly across an entire chamber of the device.
  • This has the advantage that seeding of the cells can be done for the entire chamber at once, as cells distribute evenly across the chamber and settle in the compartments and adhere to the bottom growth surface, and in specific embodiments of the device, also settle in the incisions.
  • any media or other substrates such as e.g. pharmaceuticals, antibodies, antigens, or chemical compounds, can be added to all compartments of a chamber in a single step, thereby significantly simplifying handling of the cell culture.
  • any solutions present in a chamber can be removed in a single step.
  • handling of the cell culture is simplified over the time span of the culture, e.g. from seeding the cells, up to the formation of the spheroids and investigation of the same.
  • cells adhere to the bottom surface of the two-dimensional cell culturing device described herein in a monolayer and subsequently assemble into cell spheroids of uniform size spontaneously. Surprisingly, cells spontaneously assemble into spheroids, even without non-adhesive coating of the surface of the cell culture device.
  • Rolling up of the cell layer causes detachment from the bottom surface of the cell culture device described herein and upon conversion into pellet form, anchor points to the surface are typically lost. Specifically, conversion from monolayer into pellet form is followed by condensation into a spherical shape.
  • Some cell types form spheroids by aggregation and condensation instead of a rolling up from the compartment edges, as observed in the ASCs or co-culture.
  • some cell types may also form non-detaching nodules, which are typically in the shape of half-spheroids.
  • cell spheroids are formed by spontaneous condensation in the center of the cell monolayer of a compartment, typically following detachment of the monolayer at the edge of the compartment.
  • cell spheroids formed by the method described herein are of uniform size and their size correlates to the size of the compartments in a chamber.
  • cell spheroids of ASCs comprised a diameter of about 130 pm when cultured in a two-dimensional cell culture device as described herein comprising squared compartments comprising a length and width of lmm, and comprised a diameter of about 340 pm when cultured in a two- dimensional cell culture device as described herein comprising squared
  • compartments comprising a length and width of 3mm.
  • cell spheroids produced according to the method provided herein vary in size with a standard deviation of only about 10, 20 or 30 %.
  • the variation in size is only approximately 10%, however, under conditions where cell spheroids are formed quickly, i.e. within a few days, the variation may be more than 10% since for example spheroid formation in incisions is quicker than in the compartments.
  • cells are grown in a cell culture media in the device described herein.
  • the term“cell culture medium” refers to any liquid or gel-like liquid capable of supporting the growth of cells in an in vitro environment.
  • Cell culture media generally comprise an
  • culture media used herein are composed of a complement of amino acids, vitamins, inorganic salts, glucose, and serum as a source of growth factors, hormones, and attachment factors. Serum may also be substituted with albumin alone or in combination with growth factors. In addition to nutrients, the medium ideally also helps maintain pH and osmolality.
  • the cell culture medium may be a natural or an artificial cell culture medium. Specifically, natural media consist solely of naturally occurring biological fluids and are very useful and convenient for a wide range of animal cell culture. Specific examples of natural cell culture media are for example plasma and serum. Artificial or synthetic media are prepared for example by adding nutrients (both organic and inorganic), vitamins, salts, 0 2 and C0 2 gas phases, serum proteins, carbohydrates, cofactors.
  • artificial cell culture media can be prepared for immediate cell survival, prolonged survival, indefinite growth or specialized functions.
  • cell culture media capable of supporting prolonged survival of cells are used.
  • Such media specifically comprises a balanced salt solution supplemented with various formulations of organic compounds and/or serum.
  • a specific example of media used with the method described herein is serum containing media.
  • fetal bovine serum is the most common supplement in animal cell culture media. It is used as a low-cost supplement to provide an optimal culture medium.
  • Serum provides carriers or chelators for labile or water-insoluble nutrients, hormones and growth factors, protease inhibitors, and binds and neutralizes toxic moieties.
  • a further specific example of media used with the method described herein is serum-free media.
  • presence of serum in the media can have drawbacks for the cell culture.
  • a number of serum-free media have been developed. These media are generally specifically formulated to support the culture of a single cell type, such as Knockout Serum Replacement and Knockout DMEM from Thermo Fisher Scientific for stem cells, and incorporate defined quantities of purified growth factors, lipoproteins, and other proteins, which are otherwise usually provided by the serum. These media are also referred to as‘defined culture media’ since the components in these media are known.
  • a further specific example of media used with the method described herein is chemically defined media.
  • Chemically defined medium contains contamination- free pure inorganic and organic ingredients, and may also contain pure protein additives, like growth factors.
  • constituents of this type of medium are produced in vertebrate cells, e.g. Chinese hamster ovary cells, bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, specific amino acids, and fatty acids.
  • protein-free media do not contain any protein and only contain non-protein constituents. Compared to serum- supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of any expressed product. Formulations like M EM, RPM I-1640 are protein-free and protein
  • cells are cultured in the cell culture device for a time sufficient for the formation of cell spheroids.
  • the time sufficient for the formation of cell spheroids depends on the choice of cell line and whether cells are kept in mono culture or co-culture.
  • cells are grown in the device described herein for at least 1 day.
  • cells are grown for longer than a day, specifically for 2, 3, 4, 5, or 6 days or for about a week or two weeks.
  • the cell spheroids produced according to the method provided herein can be used in a three-dimensional cell culture.
  • Cell spheroids grown in the cell culture device described herein and embedded in hydrogel or a scaffold can be used for different applications, such as for example regenerative medicine or to study organoids in an in vitro environment mimicking their endogenous surroundings.
  • cell spheroids formed for example from stem cells, or differentiated cells in co-culture with stem cells can be used as pre differentiated seedlings embedded in hydrogel for regenerative medicine
  • a three-dimensional cell culture is an artificially created environment in which cells are permitted to grow or interact with their surroundings in all three dimensions.
  • three-dimensional cell culture as used herein may comprise hydrogel embedding pre-formed cell spheroids.
  • Hydrogels can be broadly classified as either natural or synthetic materials. Hydrogels used herein may be naturally-derived, such as for example collagen, fibrin or alginate or may be synthetic, such as for example polyacrylamide or polyethylene glycol, or may be a hybrid material that combines elements of synthetic and natural polymers, such as for examples hyaluronic acid and polypeptides.
  • HACs were isolated from human femur heads obtained from joint replacements. The study was approved by the local ethics committee and patient consent was given via a consent form.
  • TERT immortalized ASC TERT immortalized ASC
  • CM Chondrocyte expansion media
  • DM EM high glucose Gibco, 41966-029
  • FCS PAN Biotech
  • 2 pg/ml amphotericin B Gibco
  • 100 pg/ml gentamicin Gibco
  • 50 pg/ml L-ascorbic acid 2-phosphate Sigma-Aldrich
  • lOmM HEPES 5 pg/ml insulin
  • 2mM L-glutamine Gibco
  • EGM-2 for ASC expansion was purchased from Lonza.
  • Hennigs differentiation medium contained DM EM high glucose (Sigma-Aldrich D6546), lx insulin-transferrin-selenium (Gibco, 41400045), 0.17 mM L-ascorbic acid 2-phosphate, 1 mM sodium pyruvate (Gibco), 0.35 mM L-proline (Sigma- Aldrich, P5607), 1.25 mg/ml bovine serum albumin (Sigma-Aldrich), lx
  • Adipose derived stromal cells (ASC/TERT1 Cat# CHS-001-0005) immortalized by ectopic expression of the catalytic subunit of human telomerase (hTERT) were provided form the Evercyte GmbH (Evercyte, Vienna, Austria,) and retrovirally infected with the sequence of the red fluorescent protein mCherry using Phoenix-Ampho cells”.
  • the petri dishes were lasered with a speed of 33,5 cm/s and in a distance of 1 or 3 mm.
  • An example of the compartmentalized plates used as cell culture devices in the present examples and the incisions is seen in Figures 12 and 13, respectively.
  • Figure 13 shows an SEM image of C02-laser generated incisions within the plastic surface of the cell culture device. The incisions go deep into the material and underneath the cull culture surface level, and are flanked by ridges on the top. Pellet formation kinetics and effects of ASC: H AC ratio.
  • Pellet sizes were measured after 5 weeks of culture using TE2000-U and N IS-Elements BP 4.20.03 (Nikon) software and data was tested for normality using D’Agostino & Pearson omnibus normality test (p ⁇ 0.0001). Statistical significance of difference in formation speed and pellet size was tested using Kruskal-Wallis and Dunn’s multiple comparison tests. Additionally time-laps imaging of pellet formation was done using Lumascope 620 and Lumaview software (Etaluma).
  • ASC Size modulation of ASC pellets.
  • thrombin 500 U I/m I ; Baxter
  • Thrombin was diluted 1:8 in CM to reduce reaction speed and fibrinogen was diluted 1:1 or 1:8 with CM containing cell pellets, resulting in highl and low-density fibrin gels respectively.
  • Following embedding pellets were cultivated for 2 weeks. Cultures were imaged twice a week and time-laps of sprouting pellets were taken for the first 3 days of culture using Lumascope 620 and Lumaview software.
  • Histology Histology samples were fixed using 4% formaldehyde overnight at 4 C and subsequently re-buffered to PBS, dehydrated in a graded series of alcohol and embedded in paraffin via xylol (Roth, Switzerland). Sections of 3-4 pm were cut and stained by AZAN for visualization of cell distribution, Alcian blue (0.3% at pH 2.5) to detect glycosaminoglycan and immunostained with collage type I I (Thermo Fisher Scientific, United States) to evaluate the differentiation.
  • Dehydration was done using an ethanol series (15%, 30%, 50%, 70%, 80%, 2x96%, 98%, 2x absolute) with 5 min incubation per step and subsequently exchanged in 3 steps to hexamethyldisilazane (Sigma-Aldrich, United States) and left to dry overnight. Samples were sputter coated with gold (Quorum) prior to imaging and were observed using a Zeiss MA-10 SEM at lOkV acceleration voltage.
  • pellet culture to grid plate culture For standard pellet culture 96 well round bottom plates were coated with poly(2-hydroxyethyl methacrylate) (poly- HEM A, Sigma, P3932). 0.5 g poly-HEMA was dissolved in 95% ethanol overnight at 38 C while shaking. 50 pi of the solution were added per well and plates were left at 37 C while shaking to evaporate ethanol for at least 8 h. ASC and ASC:HAC (50:50) pellets were created by seeding a total of 5000 cells per well and centrifuging at 650 g for 5 min, yielding pellet of a similar size to grid plates (300-350pm). Cultures on grid plates were seeded at 1 x 106 cells per plate. Cell samples before seeding and pellets after 3 and 5 weeks of cultivation, samples were taken for qRT-PCR and histology.
  • poly- HEM A poly(2-hydroxyethyl methacrylate)
  • RNA isolation and qRT-PCR Spheroids and pellets were lysed in RLT lysis buffer containing 10 pl/ml b -mercaptoethanol, frozen and kept at 80 until further processing. RNA was isolated using RNeasy Micro Kit (Qiagen) according to manufacturer protocol, eluting with 14 pi UltraPure DEPC-treated water
  • RNA concentrations were measured using NanoDrop 2000c
  • RNA samples were subsequently reverse transcribed using iScript cDNA synthesis kits (Bio-Rad) according to manufacturer protocol using Primus 25 thermal cycler (MWG Biotech).
  • qPCR was performed using SensiMix I I probe kit and TaqMan probes (20pl reactions) for Collal (Applied Biosystems, Hs00164004 ml), Col2al (Eurogentec, forward: 5’-GCC-TGG-TGT- CAT-GGG-TTT-3’, reverse: 5’-GTC-CCT-TCT-CAC-CAG-CTT-TG-3’, probe: 5’-AAA- GGT-GCC-AAC-GGT-GAG-CCT-3’), and H PRT1 (Applied Biosystems,
  • Hs02800695___ml housekeeping gene on a 7500 Fast Real-Time PCR system (Applied Biosystems). Data was analyzed using D A CT - method and are displayed as fold change mean ⁇ SD.
  • ASC are a promising cell source for tissue regeneration, including regeneration of damaged cartilage, due to them being easily available in high cell numbers from subcutaneous fat, while leading to less donor site morbidity, than the isolation of bone marrow derived stem cells. Therefore, we assessed the ability of ASC to self-assemble into micro-mass pellets, when seeded on
  • Fig. 1 depicts the results of pellet generation via 1 mm and 3 mm grid plates. Generated pellet sizes were statistically significantly different (p ⁇ 0.0001) with mean diameters of 134.4 pm (1 mm, Fig. lc) and 340.9 pm (3 mm, Fig. Id). Pellets exhibited only small variability in size, with standard deviations around 10% of pellet size or less (1 mm: 13.49 pm, 3 mm: 24.43 pm).
  • ASC:FIAC cultures were not significant, but still showed a trend to be slightly faster in pellet formation then ASC cultures.
  • pellets of different sizes and composition were embedded into low- and high-density fibrin. All conditions showed sprouting behavior, however it varied strongly between conditions (Fig. 7). Pellets embedded into high density fibrin showed minimal outgrowth after two weeks of culture, while embedding into low density gels yielded a strong sprouting already in the first days after embedding. If pellets were in proximity to each other an increased amount of directed sprouting could be observed. Small pellets exhibited similar behavior, especially in the earlier days of culture, however as cells became more disseminated, they take on a rounder morphology. Co-culture pellets performed the most invasive and while in other conditions a core remained after cellular outgrowth, it was barely visible in this condition. This was also easily observable in fluorescence, where no compact region of fluorescence remained. These differences in behavior could also easily be visualized using time-lapse imaging.
  • Example 5 Standard pellet culture vs. grid plates
  • Pellet culture is one of the standard 3D culturing systems used for differentiation of MSC and re-differentiation of chondrocytes.
  • drawbacks of this method are high time and reagent consumption. While some approaches for full automation have tried to alleviate this problem, they usually come with high cost of equipment, often not affordable for many researchers. Therefore, the goal of this study was to establish an easy to manufacture system, for self-assembly of micro-mass pellets allowing reduction of reagent usage and early read-out parameters to assess differentiation potential and therapy effects, while enabling easy harvesting of pellets for therapeutic use and end point analysis.
  • pellets When using ASC in a chondrogenic differentiation setup pellets could easily be generated in about 3 weeks, and pellet size could easily be controlled by grid size, with low size variation (standard deviation ⁇ 10%) (Fig. 1). Additionally, the generated pellets showed dark areas which have previously been reported to be associated with condensation and matrix deposition of ASC. In this setup due to reduced media consumption, reagent cost reduction up to 10 fold was achievable, assuming generation of 200 pellets with 300pm diameter (3 mm grid), with a break even point at 27 pellets generated.
  • the laser engraving method used is very flexible in regards to the cell culture vessel used as“raw-material”.
  • pellets of the same size are needed smaller vessels, thereby further reducing the amount of media consumed and lowering the break-even point further, e.g. when using 12-well plates with 1 ml medium, theoretically yielding 28 pellets, leads to a lowering of the break-even point to 7 pellets.
  • the pellet number can easily be increased by reducing grid size, while in standard pellet culture systems even if small cell numbers would be possible, reduced pellet sizes would not decrease time and space consumption during maintenance, and would only minimally reduce medium consumption.
  • compartments of grid plates Some cell types form spheroids by aggregation and condensation instead of a rolling up from the compartment edges, as observed in the ASCs or co-culture. Depending on the medium, cell type and cellular activity, some cell types form non-detaching nodules (Fig. 11). The formation of these adherent nodules in the shape of half-spheroids provides an easy-to-use and affordable platform to analyze not only cellular interactions during the aggregation phase but also the influences of drugs.
  • Example 6 Incisions made with scalpels, knives and fraises
  • Standard petri dish e.g. from Grainer, Corning
  • a scalpel e.g. from Grainer, Corning
  • a Stanley knife e.g., a Stanley knife
  • a fraise e.g., a Jerusalem knife
  • a grid pattern into the surface and compartment the surface into fields of about 9mm 2 .
  • Plates were imaged in the SEM to visualize the incisions.
  • dishes were cleaned and sterilizing with 70% alcohol and UV-illumination. Then, 1 Mio. cells (mCherry transduced ASC/TERT1) were seeded and cultivated in Hennigs chondrogenic differentiation medium containing a low dose of growth factors (lng/ml TGF b -3, and lng/ml BM P-6). Cell growth was imaged using light microscopy.
  • Figure 15 shows bright field images of a scalpel-incised
  • honeycombs the directed self-assembly of microtissues with prescribed microscale geometries. FASEB J. 21: 4005-4012. doi:10.1096/fj.07- 8710com.

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Abstract

Un procédé de production de sphéroïdes tridimensionnels de cellules, à l'aide d'un dispositif de culture cellulaire bidimensionnelle, comprend au moins une chambre ayant une surface inférieure comprenant des compartiments séparés, comprenant les étapes suivantes : i. l'introduction de milieux de culture cellulaire comprenant des cellules dans le dispositif de culture cellulaire, ii. la possibilité pour lesdites cellules de se déposer à l'intérieur des compartiments, et iii. la culture des cellules pendant une durée suffisante pour la formation de sphéroïdes cellulaires, dans au moins 80% des compartiments, au moins un sphéroïde de cellule est formé.
PCT/EP2020/063659 2019-05-17 2020-05-15 Production de sphéroïde cellulaire dans un système de culture cellulaire 2d Ceased WO2020234170A1 (fr)

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EP2617807A1 (fr) 2010-09-14 2013-07-24 Asahi Glass Company, Limited Substrat de culture
US20160324991A1 (en) 2011-09-06 2016-11-10 The Trustees Of Columbia University In The City Of New York Multiplexed In Vivo Screening Of Biological Samples
EP2759592A1 (fr) 2011-09-20 2014-07-30 Kuraray Co., Ltd. Procédé de culture de cellules adhérentes
WO2017142410A1 (fr) 2016-02-18 2017-08-24 Perkinelmer Health Sciences B.V. Moyens et procédés pour culture et analyse de cellules sphéroïdes
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